Staphylococcus aureus is key pathogen involved in nosocomial infections, frequently due to biofilm formation on indwelling devices. Once biofilms grow on these surfaces they are difficult to eradicate because the constituent cells display increased resistance to many antibiotics. This, combined with the fact that a large percentage of S. aureus isolates identified in infections now harbor resistance genes against numerous antibiotics including methicillin and vancomycin (MRSA, VRSA), makes it clear that new ways of combating staphyloccocal biofilm-associated infections need to be developed. This subproject ofthe Harvard-wide collaborative project for new MRSA treatments is focused on the development of anti-biofilm agents that target staphyloccocal biofilms. We have recently discovered two distinct mechanisms for inhibiting biofilm formation in a broad range of organisms, including S. aureus. The first mechanism is based on the finding that bacteria organize many membrane-related processes in functional microdomains that involve unusual lipids, i.e. bacteria have lipid rafts akin to those is found in eukaryotic cells. Interfering with the synthesis of these usual lipids can lead to complete elimination of biofilm formation without a lethal effect on the cells. The second discovery is that bacteria produce D-amino acids late in the life cycle of a biofilm and that these amino acids are incorporated into the peptidoglycan with the consequence that cells are released from the biofilm matrix and the biofilm is thus disassembled. While these processes have been analyzed in detail in the bacterium Bacillus subtilis, little is known about the mechanisms by which lipid synthesis inhibitors or D-amino acids prevent biofilm formation in S. aureus. To this end, we propose to: 1) Identify the lipid raft fomiation pathway in S. aureus and determine the mechanism through which anti-lipid raft agents inhibit biofilm formation, and 2) Determine the molecular mechanism through which D-amino acids affect S. aureus biofilms.